ABSTRACT Cellular cholesterol levels are tightly regulated by multiple homeostatic pathways that respond to elevations of membrane cholesterol and to enzymatically formed oxygenated cholesterol derivatives (i.e., oxysterols). Alterations in sterol sensing and trafficking pathways contribute to human inborn errors of metabolism (e.g., Niemann-Pick C disease) and to acquired disease states (e.g., atherosclerosis). Under physiological conditions, sterol-regulated transcriptional pathways act in concert to inhibit uptake of exogenous lipoproteins and suppress de novo cholesterol synthesis, resulting in half-maximal suppression of these responses within several hours. By contrast, pathophysiological cholesterol levels, such as those present in disease states, activate transcription-independent mechanisms that respond within minutes to changes in increments in membrane cholesterol. Recent studies with oxysterol enantiomers provide evidence that sterol-membrane interactions underlie these acute cholesterol homeostatic responses. We hypothesize that side-chain oxysterols serve a critical role in acute regulation of cholesterol homeostasis through direct modulation of plasma membrane lipid environment. We propose that side-chain oxysterols trigger transcription-independent regulatory pathways by disordering membrane phospholipid organization and/or increasing the accessibility of cholesterol. This hypothesis will be tested by the following Specific Aims: (1) Characterization of the mechanism by which oxysterols perturb the structure of model cholesterol-phospholipid bilayers, (2) Examination of the efect of oxysterols on cholesterol accessibility and position in physiological membranes, and (3) Examination of the mechanism by which oxysterols promote release of plasma membrane cholesterol to intracellular pools. The proposed studies wil further our understanding of how perturbations in membrane structure relay cholesterol homeostatic regulatory signals and may identify new pharmacological targets for manipulation of the cellular handling of cholesterol in disease states.